AIM: To evaluate the different visual pathways represented by the Heidelberg Engineering Perimeter flicker defined form and RareBit (magnocellular and parvocellular respectively) in different age-groups and according to the fatigue. METHODS: Totally 64 eyes of 32 healthy subjects were included in the prospective study. Each participant underwent screening—ophthalmic examination including best-corrected visual acuity, anterior and posterior segment assessment, and visual field examination with Heidelberg Edge Perimetry (HEP)-standard automated perimeter (SAP) 24-2. They were observed for 2y previously to the enrollment. This helped to define that the enrolled patients did not bear the glaucoma-developing potential. During the screening and after two years the HEP had been conducted in the standard protocol 24-2 and RareBit perimetry (RBP) in accordance with the manufacturer’s description. Participants were randomly assigned to the groups: flicker defined form (FDF)-first or RBP-first. This defined from which additional visual field test the participant started. Participants were additionally subdivided to younger and older study groups. The effect of subject variables was explored with Mann-Whitney U-test. Testing for the presence of correlations between parameters was performed using the Spearman Rank Order Correlations and confirmed by the parametric tests. For the influence of additional factors, the Kruskal-Wallis test was performed. RESULTS: The positive correlation between mean deviation (MD) and mean hit rate (MHR) and pattern standard deviation (PSD) and standard deviation of MHR (±MHR) were found in younger study group (P=0.005, r=0.481 and P=0.0074, r=0.465), whereas in the older subgroup no correlation was observed. Additionally, the randomization protocol helped in defining the role of fatigue on the HEP-FDF results. Participant for whom the HEP-FDF was performed after RareBit had significantly worse results than those for whom the HEP-FDF was first. In the younger group, the MHR and ±MHR depend from age in that group (P<0.05, r=0.43 and r=-0.57 respectively) while no age-dependent differences were found in HEP-related parameters. On the contrary in the older group the MD and PSD varies with age (P<0.05, r=0.47 and r= -0.44 respectively) while the RBP parameters remained unchanged. The questionnaire showed that participants preferred RareBit over HEP-FDF in terms of a duration time, comfort, understanding of the test procedures, and ocular pain (P<0.05). CONCLUSION: The influence of patient’s fatigue should be considered during HEP-FDF examination. An overlap hypothesis should be reevaluated after determining of other factors that affect HEP-FDF and RareBit results.